Lipid and protein kinases mediate cell signalling processes that are important for normal and disease biology. Large-scale phosphoproteomics, now routine in many mass spectrometry (MS) laboratories, should allow the quantification of signalling without a preconception of the routes within the network that may be active. Several thousand phosphorylation sites can now be measured with high accuracy by the use of quantitative techniques based on MS (Thingholm et al, Proteomics 9, 1451 (March 2009)).
Since, by definition, each phosphorylation site is the result of a kinase activity (opposed by a phosphatase activity), it should in theory be possible to use phosphoproteomics data to obtain an estimate of activity for each kinase expressed in the system under investigation (Cutillas & Jorgensen, Biochem J 434 (March 2011)). This would entail measuring known kinase substrates (i.e., specific phosphorylation sites) which could then be taken as markers of activities of such kinases. However, using phosphoproteomics data to infer the activities of kinases is not straightforward. Databases of substrate-kinase relationships are publically available and, although not comprehensive, a subset of the sites quantifiable by large-scale phosphoproteomics is represented in these databases. The challenge in using this information is that several different kinases may phosphorylate the same substrates and proteins phosphorylated in one cell type may not be expressed or be poor substrates in others. In addition, the dynamic nature of protein phosphorylation means that this modification can quickly change during the course of an experiment and variables difficult to control such as the circadian clock, cell confluence and shear stress introduced as a result of handling cell cultures can all affect protein kinase activity, thus contributing to noisy phosphoproteomics data. Thus because of stochastic effects, a phosphoproteomics experiment may show inconsistent levels of phosphorylation of the known substrate markers of a given kinase activity.
There is therefore a need in the art for a method to reliably infer protein kinase activity based on MS-based phosphoproteomics.